Many of the currently used chemotherapy agents are members of a class of drugs referred to as anti-metabolites. One type of such anti-metabolites are molecules that block or subvert one or more of the metabolic pathways involved in DNA synthesis by mimicking naturally occurring nucleic acid building blocks. Many of this type of anti-metabolites are nucleoside analogs (NAs) (wherein a nucleoside is the unphosphorylated form of a nucleotide). These NAs usually do not possess any therapeutic activity (and thus are properly termed prodrugs) and rely on their conversion, for the most part to the triphosphorylated form, to become active (prodrug to drug metabolism).
The efficiency of conversion from the administered nucleoside to the active triphosphorylated form determines the efficacy of these types of prodrugs. The serial phosphorylation of NAs to their triphosphorylated metabolite, via monophosphate and diphosphate intermediates, is catalyzed by human cellular kinases, with deoxycytidine kinase (dCK) playing a major role. dCK transfers a phosphoryl group from ATP (or other triphosphorylated nucleotides) to deoxycytidine (dC). dCK is required for the phosphorylation of numerous nucleoside analogs including AraC (1-β-D-arabinofuranosylcytosine; Cytarabine), dFdC (2′,2′-difurodeoxycytidine; Gemcitabine), FaraA (2-fluoro-9-β-D-arabinosyladenine; Fludarabine) and 2CdA (2-chlorodeoxyadenosine, Cladribine) (Van Rompay, et al., 2000, Pharmacol. Ther. 87:189-98). Therefore, the activity of dCK is one of the factors that determine the sensitivity of leukemias and solid tumors to deoxynucleoside toxicity and hence, therapy (Stegmann et al., 1995, Blood 85:1188-94; Lotfi et al., 1999, Clin. Cancer Res. 5:2438-44; Kakihara et al, 1998, Leuk. Lymphoma 31:405-9; Bergman et al., 1999, Biochem. Pharmacol. 57:397-406; Goan et al, 1999, Cancer Res. 59:4204-7).
The critical function of dCK for the efficacy of prodrug chemotherapy is evident from direct correlation between the enzyme's activity and drug sensitivity in tumor cell lines (Hapke et al., 1996, Cancer Res. 56:2343-7). Cells lacking dCK activity are resistant to a variety of drugs, including ara-C, cladribine, fludarabine and gemcitabine (Owens et al., 1992, Cancer Res. 52:2389-93; Ruiz van Haperen et al., 1994, Cancer Res. 54:4138-43) and drug sensitivity to ara-C can be restored by expressing finctional dCK protein in cells that do not express this enzyme or in which only mutationally-inactivated forms thereof are expressed (Stegmann et al., 1995, Blood 85:1188-94). Moreover, in vivo studies conducted on animal tumors using gemcitabine showed that increased dCK activity, mediated by dCK gene transfer, results in enhanced accumulation and prolonged elimination kinetics of gemcitabine triphosphate, and ultimately, to a better tumor response to the prodrug (Blackstock et al., 2001, Clin. Cancer Res. 7:3263-8).
More efficient prodrug-to-drug conversion, i.e. nucleoside analog (NA) to NA-triphosphate, would greatly increase the potency of such prodrugs and reduce undesired side effects common in chemotherapeutic treatments (due at least in part to higher concentrations of the drug needed to achieve a therapeutic result, with concomitant toxicity to normal cells and tissues). Higher concentrations of the active metabolites of nucleoside analog prodrugs, particularly in the cancer cells themselves, would result in a better therapeutic index for these prodrugs. In addition, some tumor cells develop resistance to chemotherapeutic agents that are administered as prodrugs and activated by enzymes expressed in target tumor cells, by reducing or eliminating expression of the gene or genes encoding the enzymatic activity. Resistance arising from such down-regulation of cellular gene expression could be overcome by targeted delivery of the enzyme directly to the tumor cell. Thus, there is a need for more efficient enzymes and for methods of targeting those enzymes specifically to cancer cells.